Serotonin transporter (SLC6A4; SERT) represents a primary mechanism to regulate 5-HT availability in the gut mucosa. A large body of evidence supports linkage of SERT to various GI disorders, however, the mechanisms are not well understood. Our recent studies and preliminary data demonstrated an entirely novel role of SERT and intracellular 5-HT in the activation of Aryl hydrocarbon receptor (AhR), a newly recognized IBD susceptibility gene. Interestingly, SERT KO mice fed with AhR agonist (?-naphthoflavone) showed impaired induction of CYP1A1, the canonical AhR gene target. Our preliminary data further showed that SERT was essential for the maintenance of healthy gut microbiota, as deletion of SERT in mice was associated with a reduction in actinobacteria, and altered community structure that may affect the availability of ligands known to activate AhR. Since AhR pathways regulate gut immunity, the decrease in SERT may contribute to the pathophysiology of intestinal inflammation by suppressing basal and agonist-induced AhR activity. However, the mechanisms linking this novel paradigm of the role of serotonergic machinery in dysbiosis and agonist induced AhR activation are not known. Interestingly, dietary AhR ligands such as those present in cruciferous vegetables have protective roles in ameliorating intestinal inflammation. However, a decrease in SERT expression associated with inflammation may dampen their effects and reduce effectiveness in the course of IBD. We hypothesize that SERT-mediated uptake of 5-HT is crucial for the activation of AhR in response to dietary ligands. We also hypothesize that agents which activate SERT and/or counteract its down regulation will confer novel anti-inflammatory effects via AhR dependent mechanisms. Proposed studies in Specific Aim 1 will: a) investigate cell specific mechanisms by which 5-HT activates intestinal AhR utilizing mouse and human enteroids; b) examine whether loss of SERT renders resistance to the beneficial effects of dietary AhR ligands in TNBS ileitis model; and; c) elucidate the effects of microbiota in the activation of AhR pathways utilizing fecal transfer and investigate the link between SERT and the ability of microbiota to produce AhR ligands. Given that SERT is consistently shown to be decreased in all models of inflammation and patients with IBD, proposed studies in Specific Aim 2 will examine the efficacy of natural AhR ligands present in the diet in preventing the onset of gut inflammation, when combined with agents that upregulate SERT, such as probiotic Bifidobacteria breve. In addition, the role of SERT upregulation on mechanisms of AhR activation will be investigated utilizing state-of-the-art mouse model of epithelial cell- specific inducible overexpression of SERT. Outcome of the proposed studies should define the molecular mechanisms by which 5-HT activates AhR and should establish this novel link of the host serotonergic machinery with gut inflammation via AhR and gut microbiota/metabolites. These studies should also establish the beneficial role of SERT up regulation as a novel interventional strategy for IBD.